This proposal is directed toward understanding the molecular basis of a human metabolic disorder of carbohydrate metabolism, hereditary fructose intolerance (HFI), as well as its causes, distribution, and biochemical and physiological effects. These studies will directly test hypotheses that may explain the normal gluconeogenesis that HFI patients exhibit in the absence of fructose, and therefore, may lead to a better understanding of glucose homeostasis in humans. The specific aims of the proposed investigations are: 1) the identification of mutations in the DNA from individuals with HFI in the American population, and 2) the determination of the biochemical roots of this disorder by examination of both the normal enzyme, aldolase B, and the enzymes produced from HFI alleles containing missense mutations. First, the polymerase chain reaction (PCR) will be employed to amplify DNA from blood samples of American HFI patients for the identification of known mutations by allele specific oligonucleotide hybridization (ASO), direct PCR analysis, or amplification refractory mutation (ARM) analysis. Second, for those HFI subjects who do not carry any of the 21 known mutations, similar PCR amplification of several gene fragments encoding the entire aldolase B sequence will be used for identification of new mutations. These mutations will be identified by direct sequence determination using a third primer in PCR assisted cycle sequencing. Segregation analysis of any new mutations will give genetic evidence that these mutations cause HFI. Third, site-directed mutagenesis will be performed to generate mutant enzymes containing HFI missense mutations. These HFI enzymes will be expressed and purified as fusion proteins to glutathione-S-transferase (GST). Fourth, structural and functional analyses will determine if the HFI enzymes have any residual activity. Fifth, the enzymology of normal aldolase B will be examined by measurement of the levels of several covalent reaction intermediates (Schiff base, enamine, etc.) by chemical trapping and the rates of their formation and interconversion (Schiff base, carbon-carbon bond cleavage, proton abstraction) by rapid quenching during single turnover experiments. Lastly, stable HFI enzymes will be examined in similar experiments to assess the nature of the biochemical defects that causes HFI.